Due to the proliferation of antibiotic resistant microbes, the recent discoveries of microbial pathogens mediating common ailments, and to shifts if worldwide demographics, there is a critical need for bioactive compounds with novel activities. Prior natural product discovery approaches have apparently tapped only a small fraction of microbial diversity for bioactive metabolites. In our Phase I work we designed F- factor based cloning vectors and engineered Streptomyces host strains to efficiently capture and express bioactive compounds encoded on large (40 kb) DNA fragments captured directly from uncultivated microbes. In this Phase II work we propose to optimize the vectors to accommodate DNA fragments of up to 300 kb and to generate in Streptomyces expression libraries exceeding 10/7 primary clones. These clone libraries will be screened for bioactivity and for genes encoding known pathway homologues at rates of up to 100,000 clones per second. This work will lead to the rapid discovery of new drug candidates with potential utility in a wide range of pharmaceutical applications. PROPOSED COMMERCIAL APPLICATIONS: The proposed research will optimize recombinant vector and host systems for high throughput expression screening of novel small molecules derived from uncultivated microbes. These molecules may be developed into new lead compounds with antibiotic, anti-cancer, antiviral and other pharmaceutically significant bioactive properties.